The overall objective of this project is to understand in detail how a eukaryotic chromosome replicates and segregates. To this end a 185 kilobase (kb) circular derivative of yeast chromosome III has been cloned and a restriction map constructed. Since the average spacing of replication origins, measured by electron microscopy of replicating chromsomal DNA, is 36 kb, this chromosome is expected to have 5-7 origins of replication. This chromosome and derivatives of it will be used to study the location, structure and efficiency of replication origins, to study the temporal sequence of replication, and to identify genes whose products are necessary for the replication of this chromosome. This particular proposal is addressed to the following issues. Sequences on chromosome III which are good candidates for replication origins have been cloned and mapped. These autonomously replicating sequences (ARS's) allow the extrachromosomal replication of plasmids in yeast. These sequences will be studied to determine whether replication initiates at these sites in vivo, whether these sequences are necessary for normal chromosome function and what DNA sequences are essential for ARS function. Genes whose products interact with ARS's will be identified using a novel selection scheme. Cloned fragments of chromosome III will be used in several experiments designed to determine whether there is a fixed temporal order of replication on the chromosome. If so, then sequences in which the information that specifies the temporal order will be identified. These studies should greatly increase our understanding of a fundamental cellular process, DNA replication. These questions cannot be approached in higher eukaryotes at the present time because their chromosomal DNA's are too large to isolate without breakage and because no autonomously replicating chromosomal sequences have been identified which function in the cells from which they were isolated. Knowledge we gain from the yeast system should be applicable to higher cells, and may help lead to an understanding of the basis of unscheduled DNA synthesis, which is characteristic of malignant cells.